Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

The present invention relates to the detection of HIV by amplification and
PCR-based methods.

Claims:

1. A process for the detection of at least one HIV target, comprising the
step of producing at least one amplicon by means of at least two
oligonucleotides,wherein said oligonucleotides are primers which are
suitable for use in the specific amplification of at least one reference
template sequence selected from the group consisting of:positions
4281-4436 (SEQ ID NO: 2) of the K03455 HIV1-M reference isolate; and the
fragments of SEQ ID NO: 2 selected from positions 4281-4429 (SEQ ID NO:
306), 4283-4429 (SEQ ID NO: 307), 4283-4431 (SEQ ID NO:308) of said
HIV1-M isolate;positions 4336-4491 (SEQ ID NO: 4) of the L20587 HIV1-O
reference isolate, and the fragments of SEQ ID NO: 4 selected from
positions 4336-4484 (SEQ ID NO:310), 4438-4484 (SEQ ID NO:311) of said
HIV1-.beta. isolate;positions 4889-5036 (SEQ ID NO: 5) of the M30502 HIV2
reference isolate;positions 4176-4436 (SEQ ID NO: 1) of the K03455 HIV1-M
reference isolate, and the fragment of SEQ ID NO:1 which is identical to
positions 4176-4429 (SEQ ID NO:305) of said HIV1-M isolate; andpositions
4231-4491 (SEQ ID NO: 3) of the L20587HIV1-O reference isolate, and the
fragment of SEQ ID NO:3 which is identical to positions 4231-4484 (SEQ ID
NO: 309) of said HIV1-.beta. isolate;said reference template sequences
sharing the specific technical feature of being suitable references to
construct and produce primers which allow for a real-time quantitative
multiplex detection of at least one of the HIV1-M, HIV1-O, HIV2-A and
HIV2-B subtypes;and optionally, comprising the step of detecting said
amplicon by means of at least one probe.

3. The process of claim 2, wherein said process is carried out with at
least one primer selected from the group consisting of:SEQ ID NO: 9, 12,
14, 15, 24, 52, 82.

4. The process of claim 2, wherein said process is carried out with at
least one probe selected from the group consisting of:SEQ ID NO: 91, 100,
109, 127, 136.

5. The process of claim 2, wherein said process is carried out with at
least one primer selected from the group consisting of:SEQ ID NO:9, 12,
14, 15;and with at least one primer selected from the group consisting
of:SEQ ID NO: 24, 52, 82;and, optionally, with at least one probe
selected from the group consisting of:SEQ ID NO: 91, 100, 109, 118, 127,
136.

6. The process of claim 1, wherein said reference template sequence is SEQ
ID NO:4, and wherein said process is carried out with at least one
oligonucleotide selected from the group consisting of:SEQ ID NO: 148,
157, 167, 170, 179, 189, 190 to 194.

7. The process of claim 6, wherein said process is carried out with at
least one primer selected from the group consisting of:SEQ ID NO: 148,
157, 167, 170, 179, 189.

8. The process of claim 6, wherein said process is carried out with at
least one probe selected from the group consisting of:SEQ ID NO: 190 to
194.

9. The process of claim 6, wherein said process is carried out with at
least one primer selected from the group consisting of:SEQ ID NO: 148,
157, 167;and with at least one primer selected from the group consisting
of:SEQ ID NO: 170, 179, 189;and, optionally, with at least one probe
selected from the group consisting of:SEQ ID NO: 190 to 194.

10. The process of claim 1, wherein said reference template sequence is
SEQ ID NO: 5, and wherein said process is carried out with at least one
oligonucleotide selected from the group consisting of:SEQ ID NO: 221,
249, 276, 287, 296, 297 to 303.

11. The process of claim 10, wherein said process is carried out with at
least one primer selected from the group consisting of:SEQ ID NO: 221,
249, 276, 287, 296.

12. The process of claim 10, wherein said process is carried out with at
least one probe selected from the group consisting of:SEQ ID NO: 297 to
303.

13. The process of claim 10, wherein said process is carried out with at
least one primer selected from the group consisting of:SEQ ID NO: 221,
249;and with at least one primer selected from the group consisting
of:SEQ ID NO: 276, 287, 296;and, optionally, with at least one probe
selected from the group consisting of:SEQ ID NO: 297 to 303.

14. The process of claim 1, wherein said reference template sequence is
SEQ ID NO: 1, and wherein said process is carried out with at least one
oligonucleotide selected from the group consisting of:SEQ ID NO: 6, 24,
52, 82, 91, 100, 109, 127, 136.

15. The process of claim 14, wherein said process is carried out with at
least one primer selected from the group consisting of:SEQ ID NO: 6, 24,
52, 82.

16. The process of claim 14, wherein said process is carried out with at
least one probe selected from the group consisting of:SEQ ID NO: 91, 100,
109, 127, 136.

17. The process of claim 14, wherein said process is carried out with at
least one primer selected which is:SEQ ID NO: 6;and with at least one
primer selected from the group consisting of:SEQ ID NO: 24, 52, 82;and,
optionally, with at least one probe selected from the group consisting
of:SEQ ID NO: 91, 100, 109, 118, 127, 136.

18. The process of claim 1, wherein said reference template sequence is
SEQ ID NO: 3, and wherein said process is carried out with at least one
oligonucleotide selected from the group consisting of:SEQ ID NO: 139,
170, 179, 189, 190 to 194.

19. The process of claim 18, wherein said process is carried out with at
least one primer selected from the group consisting of:SEQ ID NO: 139,
170, 179, 189.

20. The process of claim 18, wherein said process is carried out with at
least one probe selected from the group consisting of:SEQ ID NO: 190 to
194.

21. The process of claim 18, wherein said process is carried out with at
least one primer which is:SEQ ID NO: 139;and with at least one primer
selected from the group consisting of:SEQ ID NO: 170, 179, 189;and,
optionally, with at least one probe selected from the group consisting
of:SEQ ID NO: 190 to 194.22.

23. An Amplicon obtainable by implementation of the process of claim 1 on
a HIV-containing sample.

24. An amplification composition comprising at least one amplicon of claim
23.

25. A polynucleotide suitable for use as a reference template sequence in
the design of primers that can be used in multiplex to cover at least
HIV1-M, HIV1-O, HIV2-A and HIV2-B in a single amplification run while
still offering a real time quantitative amplification thereof, said
reference template polynucleotide being selected from the group
consisting ofpositions 4281-4436 (SEQ ID NO: 2) of the K03455 HIV1-M
reference isolate; and the fragments of SEQ ID NO: 2 selected from
positions 4281-4429 (SEQ ID NO: 306), 4283-4429 (SEQ ID NO: 307),
4283-4431 (SEQ ID NO: 308) of said HIV1-M isolate;positions 4336-4491
(SEQ ID NO: 4) of the L20587 HIV1-O reference isolate; and the fragments
of SEQ ID NO: 4 selected from positions 4336-4484 (SEQ ID NO: 310),
4338-4483 (SEQ ID NO: 311) of said HIV1-.beta. isolate;positions
4889-5036 (SEQ ID NO: 5) of the M30502 HIV2 reference isolate;positions
4176-4436 (SEQ ID NO: 1) of the K03455 HIV1-M reference isolate, and the
fragment of SEQ ID NO:1 which is identical to positions 4176-4429 (SEQ ID
NO: 305) of said HIV1-M isolate; andpositions 4231-4491 (SEQ ID NO: 3) of
the L20587 HIV1-O reference isolate, and the fragment of SEQ ID NO:3
which is identical to positions 4231-4484 (SEQ ID NO: 309) of said HIV1-O
isolate.

29. The oligonucleotide of claim 26, selected from the group consisting
in:SEQ ID NO: 221, 249, 276, 287, 296, 297 to 299;wherein said
oligonucleotide is suitable for HIV2-A detection.

30. The oligonucleotide of claim 26, selected from the group consisting
in:SEQ ID NO: 221, 249, 276, 287, 296, 300 to 303;wherein said
oligonucleotide is suitable for HIV2-B detection.

31. The oligonucleotide of claim 26, wherein said oligonucleotide is
fluorescently labelled.

32. A set of oligonucleotides suitable for HIV detection, comprising at
least two oligonucleotides selected from two different groups among the
following groups:the group consisting in SEQ ID NO: 6, 9, 12, 14, 15;the
group consisting in SEQ ID NO: 24, 52, 82;the group consisting in SEQ ID
NO: 91, 100, 109, 118, 127, 136;wherein said set of oligonucleotides is
suitable for HIV1-M detection.

33. A set of oligonucleotides suitable for HIV detection, comprising:at
least one oligonucleotide selected from the group consisting in SEQ ID
NO: 139, 148, 157, 167; and/orat least one oligonucleotide selected from
the group consisting in SEQ ID NO: 170, 179, 189; and/orat least one
oligonucleotide selected from the group consisting in SEQ ID NO: 190 to
194;wherein said set of oligonucleotides is suitable for HIV1-O
detection.

34. A set of oligonucleotides suitable for HIV detection, comprising:at
least one oligonucleotide selected from the group consisting in SEQ ID
NO: 221, 249; and/orat least one oligonucleotide selected from the group
consisting in SEQ ID NO: 276, 287, 296; and/orat least one
oligonucleotide selected from the group consisting in SEQ ID NO: 297 to
299;wherein said set of oligonucleotides is suitable for HIV2-A
detection.

35. A set of oligonucleotides suitable for HIV detection, comprising:at
least one oligonucleotide selected from the group consisting in SEQ ID
NO: 221, 249; and/orat least one oligonucleotide selected from the group
consisting in SEQ ID NO: 276, 287, 296; and/orat least one
oligonucleotide selected from the group consisting in SEQ ID NO: 300 to
303;wherein said set of oligonucleotides is suitable for HIV2-B
detection.

36. A set of oligonucleotides suitable for HIV detection, comprising:at
least one oligonucleotide selected from the group consisting in SEQ ID
NO: 91, 100, 109, 118, 127, 136; and/orat least one oligonucleotide
selected from the group consisting in SEQ ID NO: 190 to 194; and/orat
least one oligonucleotide selected from the group consisting in SEQ ID
NO: 297 to 299; and/orat least one oligonucleotide selected from the
group consisting in SEQ ID NO: 300 to 303;wherein said set of
oligonucleotides is suitable for HIV multiplex detection.

37. A set of oligonucleotides of claim 32, wherein at least one of said
oligonucleotides is fluorescently labelled.

38. An amplicon obtainable by amplification from a HIV-containing sample
with a pair of primers selected from:a primer of SEQ ID NO: 9, 12, 14 or
15, and a primer of SEQ ID NO: 24, 52 or 82; ora primer of SEQ ID NO:
148, 157 or 167, and a primer of SEQ ID NO: 170, 179 or 189; ora primer
of SEQ ID NO: 221 or 249, and a primer of SEQ ID NO: 276, 287 or 296; ora
primer of SEQ ID NO: 6, and a primer of SEQ ID NO: 24, 52 or 82; ora
primer of SEQ ID NO: 139, and a primer of SEQ ID NO: 170, 179 or 189.

39. An amplicon which has a nucleotide length identical to the nucleotide
length of a polynucleotide of claim 25, and which comprises a sequence
having a percentage of nucleotide identity of at least 90% with a probe
sequence-, over the entire length of this probe sequence, wherein this
probe sequence is one of the following: SEQ ID NO: 91, 100, 109, 118,
127, 136, 190, 191, 192, 193, 194, 297, 298, 299, 300, 301, 302, 303, or
a sequence which is complementary to one of these probe SEQ ID NO:
sequences over the entire length of this probe SEQ ID NO: sequence.

40. An amplification composition comprising an amplicon of claim 38 or 39.

41. A kit for HIV diagnostics, comprising:a. at least one oligonucleotide
selected from the group consisting of SEQ ID NO:6, 8, 11, 14, 15, 24, 52,
82, 91, 100, 109, 127, 136, 139, 148, 157, 167, 170, 179, 189, 190 to
194, 221, 249, 276, 287, 296, and 297 to 303; and/orb. at least one set
of oligonucleotides of any one of claims 32-37; andc. optionally,
instructions for the use thereof.

Description:

FIELD OF THE INVENTION

[0001]The present invention relates to the field of HIV and HIV subtypes.
More precisely, the invention relates to the detection of HIV types and
subtypes, and especially to the multiplex detection of HIV types and
subtypes.

BACKGROUND ART

[0002]HIV (Human Immunodeficiency Virus) is the virus responsible for the
acquired immunodeficiency syndrome (AIDS), and belongs to the human
retrovirus family. AIDS is now considered as one of the greatest threats
to human health. An HIV-infected individual can transmit the disease,
although remain asymptomatic for years.

[0004]The HIV family comprises several types and subtypes. HIV1 viruses
can be classified according to subtypes. Examples of HIV1 sub-types
include HIV1-M and HIV1-O. Similarly, HIV2 viruses encompass various
sub-types, e.g. HIV2-A and HIV2-B.

[0005]For drug development assays, prophylaxis, as well as for treatment
of AIDS, it has now become of great importance to be able to quickly and
easily identify and quantify the group(s), type(s) and subtype(s) of HIV
viruses present in a given sample.

[0006]By HIV group, we herein understand any HIV group, irrespective of it
being known at the priority date or not. Various HIV groups are known in
the art, and are described in the corresponding literature and databases,
e.g. ncbi on the internet. Examples thereof include HIV1-M and HIV1-O.

[0007]By HIV subtype, we herein understand any HIV subtype, irrespective
of it being known at the priority date or not. Various HIV subtypes are
known in the art, and are described in the corresponding literature and
databases, e.g. ncbi on the internet.

[0008]By HIV isolate, we herein understand any HIV isolate or strain,
irrespective of it being known at the priority date or not. Various HIV
isolates are known in the art, and are described in the corresponding
literature and databases, e.g. ncbi on the internet. Some isolates are
regarded as references. Examples thereof include K03455, L20587 and
M30502.

[0009]A possible approach could rely on the development of specific
antibodies. However, in terms of sensitivity and specificity, a PCR-based
approach usually looks very promising. Also, it generally offers the
possibility to work on small samples.

[0014]Depending upon the working conditions, at least some of these prior
art primers may show a sufficient HIV1 or HIV2 specificity, thereby
allowing for a specific detection of HIV1 and HIV2. However, to the
applicant's knowledge, none of them allows for: [0015]a real-time
quantitative specific detection of said subtypes, or for [0016]a
detection of said subtypes in multiplex which would remain specific, or
for [0017]a detection of said subtypes in multiplex which would remain
quantitative, even when implemented in real-time.

[0018]Such a real-time quantitative multiplex specific detection would
however be more reliable and informative on the patient's actual
infection stage. It would thus give access to a more accurate diagnosis,
and as a consequence, would allow to more accurately balance the positive
against the deleterious effects of a given treatment. It would allow
adjusting or choosing the treatment which should be the most efficient to
the particular patient being diagnosed.

[0019]Such a real-time quantitative multiplex specific detection would
also have the advantage of being faster and easier to run, especially on
a large scale.

DESCRIPTION OF THE INVENTION

[0020]The present invention provides a process for HIV detection.

[0021]The present invention provides oligonucleotides, including primers
and probes, and sets thereof, which are suitable for the detection of
HIV.

[0022]In this respect, the present invention also relates to the field of
amplification, PCR and PCR-based methods, as well as diagnostics.

[0024]By real-time PCR, we hereby understand any PCR-based method allowing
for monitoring of fluorescence emitted during the reaction as an
indicator of amplicon production during each PCR cycle as opposed to the
endpoint detection by conventional PCR methods.

[0025]By quantitative PCR, we hereby understand any PCR-based method
allowing for the estimation of the initial amount of a given PCR target
in a given sample.

[0026]By multiplex PCR, we hereby understand any PCR reaction aiming at
the amplification of more than one target. For instance, multiplex PCR
include duplex PCR (two targets), triplex PCR (three targets), and higher
multiplex PCR. Multiplex PCR includes PCR reactions with more than one
primer pair, for instance two primer pairs. In this case, there might be
four different primers, but it is also possible for the two primer pairs
to have one primer in common, e.g. the forward primer, and to have two
distinct reverse primers. Multiplex PCR also includes PCR reactions with
a unique primer pair, but with more than one probe.

[0027]By oligonucleotide, we hereby understand any short polymer of
nucleotides, wherein nucleotides can be ribonucleotides,
deoxyribonucleotides, dideoxyribonucleotides, degenerated nucleotides,
and the like. Said oligonucleotides are preferably single-stranded. The
length of said oligonucleotides can vary, and is usually under 150
nucleotides (nt), preferably in the range of 10-100 nt, more preferably
15-60 nt, even more preferably 18-50 nt. Said oligonucleotides can bear
chemical modifications, such as tagging or marking, for instance
radioactive, fluorescent, biotinylated, dig labelling. An oligonucleotide
according to the invention can be either forward (sense) or reverse
(antisense). In addition, it should be stressed, that although preferred
functions may be mentioned in relation to some oligonucleotides according
to the present invention, it is obvious that a given oligonucleotide may
assume several functions, and may be used in different ways according to
the present invention. For example, an oligonucleotide can be used either
as a primer, or as a probe. Also, when an oligonucleotide is described as
being useful as an amplicon-targeting probe, the skilled person
understands that the complementary sequence of this oligonucleotide is
equally useful as a probe to target the same amplicon. Moreover, it is
also obvious, that any primer suitable for a multiplex assay, can also,
within the meaning of the present invention, be used in a simplex
protocol. The same applies to a primer suitable for a real-time protocol,
which can also be used in the framework of an end-point assay within the
meaning of the present invention.

[0028]Oligonucleotides according to the invention especially include PCR
primers and probes. Unless otherwise stated, nucleic acid sequences are
given in the 5' to 3' direction. Said oligonucleotides can be under many
forms, e.g. under dry state, in solution/suspension with the desired
solvent and the desired concentration. The skilled person would know,
which solvents, concentrations, storage conditions are suitable for the
oligonucleotides of the invention. In particular, the skilled person
would know how to prepare said oligonucleotides as stock solutions. The
oligonucleotides according to the invention can also assume various
degrees of purity, as can be judged by those skilled in the art, e.g. by
HPLC chromatography.

[0029]By set of oligonucleotides, we hereby understand any combination
comprising at least one oligonucleotide, preferably at least two, e.g.
2-10 oligonucleotides. Said set can thus comprise one PCR primer, or a
pair of PCR primers, or a probe, or a probe and a pair of primers. Said
oligonucleotides can be separately kept, or partially mixed, or entirely
mixed.

[0030]The notion of primer or PCR primer is known to those skilled in the
art. For example, it includes any oligonucleotide able to anneal to a
target template under suitable stringency conditions, and allowing for
polymerase strand elongation. The typical length of said primer is 15-30
nt, preferably 18, 19, 20, 21, 22, 23, 24 or 25 nt.

[0031]The notion of probe is also known to those skilled in the art. For
example, it includes any oligonucleotide able to anneal to a target
template under the desired hybridization conditions. The typical length
of said probe is 20-55 nt, preferably 15-60 nt, more preferably 20-55 nt,
more preferably 30-50 nt, more preferably 35-45 nt. Preferably, said
probe is fluorescently labelled. However, it is clear to those skilled in
the art that under certain conditions, one may use a primer as a probe
and vice-versa. Moreover, it is herein stressed that the products
according to the present invention, especially, inter alia,
oligonucleotides, are not limited to the intended use herein mentioned,
but rather are to be broadly construed, irrespective of the indicated
destination. For instance, a claim to a product (oligonucleotide) for a
particular use should be construed as meaning a product (oligonucleotide)
which is in fact suitable for the stated use. Thus, an oligonucleotide
suitable for use as a primer in a multiplex protocol is also clearly
adapted to a simplex protocol within the meaning of the present
invention.

[0032]Various formats (types) of probes, including Taqman® probes
(hydrolysis probes), molecular Beacons® (beacon probes or molecular
beacon probes), and Scorpion® probes are known in the art.

[0033]In a preferred embodiment, the probes according to the invention can
all be synthesized and used in the molecular beacon format.

[0034]The structure of molecular beacons is as follows. A short nucleotide
sequence (so-called beacon arm) which is unrelated to the target sequence
is thus covalently linked to both ends of the probe. A short unrelated
arm is thus linked in 5' of the probe, and is labelled with a fluorescent
moiety (i.e. fluorescent dye or fluorescent marker). Another but still
unrelated arm is linked to the 3' end of probe and is labelled with a
fluorescence quenching moiety. Thus, molecular beacons have a fluorophore
and a quencher at opposite ends. The 5' short arm is totally
complementary to the one in 3' so that they can anneal together, and thus
can assume a hairpin structure when unhybridized to the target in
solution. In this hairpin conformation, the quencher and the fluorescent
dye are close enough to each other to allow efficient quenching of the
fluorophore. However, when the probe encounters a target molecule,
annealing is favoured with respect to the hairpin conformation when
values of beacon arm Tm and probe Tm are suitably chosen (theoretically:
probe Tm>beacon arm Tm>primer Tm, wherein Tm is the melting
temperature of interest).The fluorophore and quencher move away from each
other and the fluorophore can then fluoresce when illuminated by suitable
light excitation. AsPCR proceeds, amplification product accumulates, and
the amount of fluorescence at any given cycle depends on the amount of
amplification product present at that time. (See e.g. Sanjay Tyagi and
Fred Russell Kramer, Nature Biotechnology 1996, volume 14, pages 303-308;
Nature Biotechnology 1998, volume 16, pages 49-53).

[0035](Remark: It is also possible to link the fluorophore at the 3' end,
while attaching the quencher at the 5' end.)

[0036]Schematically, said probe can have the following formulae (molecular
beacon format): [0037]5' Fluorophore-(arm 1)-probe-(arm2)-Quencher 3'
[0038]5' Quencher-(arm 1)-probe-(arm2)-Fluorophore 3'wherein arm1 and
arm2 can be any short nucleotide sequences, e.g. in the range of 3-10
nucleotides, preferably 5, 6, 7 nucleotides, allowing for the hair pin
structure formation under suitable stringency conditions, i.e. arm1 and
arm2 are totally complementary to anneal under the desired stringency
conditions (standard PCR stringency conditions include, for example, an
annealing temperature of 55 to 65° C. and an Mg concentration of 4
to 8 mM). However, arm1 and arm2 are unrelated to the target sequence of
the probe, i.e. the hairpin conformation resulting from the annealing
between arm1 and arm2 is essentially the only possible secondary
structure for the probe when unhybridized. The skilled person would know
how to choose such arms for a given probe.

[0041]By quencher, we herein understand any quencher known in the art.
Examples of such quenchers include Dabcyl, Dark Quencher, Eclipse Dark
Quencher, ElleQuencher, Tamra, BHQ and QSY (all of them are Trade-Marks).

[0042]The skilled person would know which combinations of dye/quencher are
suitable when designing a probe.

[0043]In a preferred embodiment according to the invention, spectral
properties of said probes can be chosen as to not interfere with each
other. In particular, when probes are used in multiplex, each single
probe can have its own fluorophore being spectrally significantly
different from each other, i.e. the absorption/emission spectra are
essentially non-overlapping. This advantageously allows for low-noise
multiplex detection for all single probes, making sure that individual
signals do not interfere with each other in detection. Examples of dyes
which can be used together in multiplex include Fam with Tamra, Fam with
Tamra with Texas Red.

[0044]According to the invention, all the provided oligonucleotides can be
either kept separately, or partially mixed, or totally mixed.

[0045]Said oligonucleotides can be provided under dry form, or solubilized
in a suitable solvent, as judged by the skilled person. Suitable solvents
include TE, PCR-grade water, and the like.

[0046]Thereafter, sequences are identified by a SEQ ID NO:

[0047]The corresponding sequences are given on the tables in the figures
appended thereto. In said tables, the standard code for degenerated
nucleotides is used. In particular: R is G or A; Y is C or T; W is A or
T.

[0048]In the given sequences, where several positions are degenerated, it
is clear to those skilled in the art that each degenerated position can
be chosen independently from each other. For example, RY can be GC, GT,
AC, AT, combinations and mixtures thereof. Thus, SEQ ID NO: 52 may be any
one of SEQ ID NO: 25 to 51 (see table) or combinations or mixtures
thereof, etc.

[0049]In addition, in said tables, d indicates a degenerated
oligonucleotide; e denotes an expanded oligonucleotide (i.e. a lengthened
version of another oligonucleotide); and 1 designates a loop
oligonucleotide.

[0050]By loop oligonucleotide, we hereby understand any oligonucleotide,
whose 5' end has been modified by addition of a few nucleotides
(generally 3, 4, 5, 6 or 7 nt) so as to be complementary to the 3' end of
said any oligonucleotide. Thus, said loop oligonucleotide has the
advantageous feature of being able to adopt a loop conformation under
given stringency conditions. This property is extremely advantageous in
increasing the specificity and sensitivity in a PCR protocol, in
particular in a multiplex protocol, by avoiding interactions between
primers, or between primers and probes.

[0051]The present invention provides a process for the detection of HIV.
In one aspect, the invention provides a process for the detection of at
least one HIV target, comprising the step of producing at least one
amplicon by means of at least two oligonucleotides, [0052]wherein said
oligonucleotides are suitable for use in the specific amplification of at
least one reference template sequence selected from the group consisting
of: [0053]positions 4281-4436 (SEQ ID NO: 2) of the K03455 HIV1-M
reference isolate; and the fragments of SEQ ID NO: 2 selected from
positions 4281-4429 (SEQ ID NO: 306), 4283-4429 (SEQ ID NO: 307),
4283-4431 (SEQ ID NO: 308) of said HIV1-M isolate; [0054]positions
4336-4491 (SEQ ID NO: 4) of the L20587 HIV1-O reference isolate, and the
fragments of SEQ ID NO: 4 selected from positions 4336-4484 (SEQ ID NO:
310), 4438-4484 (SEQ ID NO:311) of said HIV1-β isolate;
[0055]positions 4889-5036 (SEQ ID NO: 5) of the M30502 HIV2 reference
isolate; [0056]positions 4176-4436 (SEQ ID NO: 1) of the K03455 HIV1-M
reference isolate, and the fragment of SEQ ID NO:1 which is identical to
positions 4176-4429 (SEQ ID NO: 305) of said HIV1-M isolate; and
[0057]positions 4231-4491 (SEQ ID NO: 3) of the L20587 HIV1-O reference
isolate, and the fragment of SEQ ID NO:3 which is identical to positions
4231-4484 (SEQ ID NO: 309) of said HIV1-β isolate.

[0058]The reference template sequences hence correspond to isolated
fragments of a determined HIV isolate (i.e. the fragment which is
identical to the sequence extending from the indicated positions). The
oligonucleotides suitable for use in the specific amplification of at
least said reference template sequence are hence selected to target this
reference template sequence, in such a location that they would lead to
the amplification of this reference template sequence under the form of
an isolated fragment.

[0059]In another aspect, the invention provides a process for the
detection of at least one HIV target, comprising the step of producing at
least one amplicon by means of at least two oligonucleotides,
[0060]wherein said oligonucleotides are `quantitative friendly` and/or
`multiplex-friendly` primers which are suitable for use in the specific
amplification of at least one reference template sequence selected from
the group consisting of: [0061]positions 4281-4436 (SEQ ID NO: 2) of the
K03455 HIV1-M reference isolate; and the fragments of SEQ ID NO: 2
selected from positions 4281-4429, 4283-4429, 4283-4431 of said HIV1-M
isolate (SEQ ID NO: 306, 307 and 308); [0062]positions 4336-4491 (SEQ ID
NO: 4) of the L20587 HIV1-O reference isolate, and the fragments of SEQ
ID NO: 4 selected from positions 4336-4484, 4438-4484 of said HIV1-β
isolate (SEQ ID NO: 310 and 311); [0063]positions 4889-5036 (SEQ ID NO:
5) of the M30502 HIV2 reference isolate; [0064]positions 4176-4436 (SEQ
ID NO: 1) of the K03455 HIV1-M reference isolate, and the fragment of SEQ
ID NO:1 which is identical to positions 4176-4429 (SEQ ID NO: 305) of
said HIV1-M isolate; and [0065]positions 4231-4491 (SEQ ID NO: 3) of the
L20587 HIV1-O reference isolate, and the fragment of SEQ ID NO:3 which is
identical to positions 4231-4484 (SEQ ID NO: 309) of said HIV1-β
isolate.

[0066]In another aspect, the invention provides a process for the
detection of at least one HIV target, comprising the amplification of at
least one reference template sequence selected from the group consisting
of: [0067]positions 4281-4436 (SEQ ID NO: 2) of the K03455 HIV1-M
reference isolate; and the fragments of SEQ ID NO: 2 selected from
positions 4281-4429, 4283-4429, 4283-4431 of said HIV1-M isolate;
[0068]positions 4336-4491 (SEQ ID NO: 4) of the L20587 HIV1-O reference
isolate, and the fragments of SEQ ID NO: 4 selected from positions
4336-4484, 4438-4484 of said HIV1-β isolate; [0069]positions
4889-5036 (SEQ ID NO: 5) of the M30502 HIV2 reference isolate;
[0070]positions 4176-4436 (SEQ ID NO: 1) of the K03455 HIV1-M reference
isolate, and the fragment of SEQ ID NO:1 which is identical to positions
4176-4429 of said HIV1-M isolate; and [0071]positions 4231-4491 (SEQ ID
NO: 3) of the L20587 HIV1-O reference isolate, and the fragment of SEQ ID
NO:3 which is identical to positions 4231-4484 of said HIV1-0 isolate.

[0072]In a preferred embodiment, said process comprises the step of
detecting said amplicon by means of at least one probe.

[0073]The term amplicon is known to those skilled in the art. By amplicon,
we herein understand any amplification product.

[0074]Amplification is known in the art, and can be any process involving
at least one amplification step, in particular at least one PCR or a
PCR-based amplification step.

[0075]By `multiplex-friendly` oligonucleotide, we herein understand any
oligonucleotide which can be successfully used in a multiplex (PCR)
protocol. In particular, `multiplex-friendly` oligonucleotides allow
specific and sensitive results in a multiplex protocol.

[0076]By `quantitative friendly` oligonucleotide, we herein understand any
oligonucleotide which can be successfully used in a quantitative (PCR)
protocol. In particular, `quantitative-friendly` oligonucleotides allow
specific, sensitive and quantitative results.

[0077]A sequence complementary to another sequence is herein meant as a
sequence which is complementary to said other sequence over the entire
length of this other sequence.

[0078]Although the process according to the present invention can
advantageously be carried out in the framework of a multiplex protocol,
it is also clearly possible to carry it out as a simplex protocol, for
example a simplex end-point or qualitative protocol.

[0079]By reference template sequence, we herein understand any template
sequence which can be used as a reference for alignment. For example,
some genomes are considered as reference genome. Sequence alignment is
known in the art. Advantageously according to the invention, SEQ ID NO: 1
to 5 and the above-mentioned fragments thereof are reference template
sequences sharing the specific technical feature of being suitable
references to construct and produce primers which allow for a
quantitative detection of at least one of the HIV1-M, HIV1-O, HIV2-A and
HIV2-B subtypes. Said reference template sequences are suitable
references to construct and produce primers which allow for: [0080]a
real-time quantitative detection of at least one of said HIV subtypes,
[0081]a multiplex detection of at least one of said HIV subtypes, [0082]a
real-time quantitative multiplex detection of at least one of said HIV
subtypes.

[0087]Thus, the process according to the invention advantageously allows
the specific and sensitive detection of the main HIV groups and/or types
and/or subtypes, and of possibly virtually any HIV groups and/or types
and/or subtypes, by a real-time quantitative and/or multiplex
amplification protocol. Examples of such groups and/or types and/or
subtypes covered by the process according to the present invention
include HIV1-M subtypes A (A1 and A2), B, C, D, F (F1 and F2), G, H, J
and K, but also the recombinant forms AE, AG, AB, DF, BC, CD, BF and BG,
and also U (highly divergent). Further examples thereof comprises HIV2
subtypes A, B.

[0088]The invention hence provides a HIV detection process by nucleic by
acid amplification which: [0089]can be group- and/or type- and/or
subtype-specific, [0090]is quantitative, and more particularly allows for
a real-time quantitative HIV detection, [0091]can be implemented in
multiplex while still remaining specific, and can even be implemented in
a (possibly real-time) quantitative multiplex amplification protocol.

[0092]Thus, the process according to the invention can advantageously
facilitate diagnostics procedures by differentiating-HIV1-M and HIV1-0
groups from HIV2 group and covering a very broad spectrum of HIV types
and/or sub-types using a single procedure.

[0093]In one aspect of the invention, said reference template sequence is
SEQ ID NO: 2, from the K03455 HIV1-M reference isolate, or a fragment of
SEQ ID NO: 2 selected from positions 4281-4429, 4283-4429, 4283-4431.

[0094]In one embodiment, said reference template sequence is SEQ ID NO: 2,
from the K03455 HIV1-M reference isolate, or one of said fragments of SEQ
ID NO: 2, and said process is carried out with at least one
oligonucleotide selected from the group consisting of: [0095]SEQ ID NO:
9, 12, 14, 15, 24, 52, 82, 91, 100, 109, 118, 127, 136, and the sequences
which are complementary to one of SEQ ID NO: 91, 100, 109, 118, 127, 136
over the entire length of this SEQ ID NO:

[0096]In a preferred embodiment, said process is carried out with at least
one primer selected from the group consisting of: [0097]SEQ ID NO: 9,
12, 14, 15, 24, 52, 82.

[0098]In another embodiment, said process is carried out with at least one
probe selected from the group consisting of: [0099]SEQ ID NO: 91, 100,
109, 118, 127, 136, and the complementary sequences thereof.

[0100]In a more preferred embodiment, said process is carried out with at
least one primer selected from the group consisting of: [0101]SEQ ID
NO:9, 12, 14, 15.and with at least one primer selected from the group
consisting of: [0102]SEQ ID NO: 24, 52, 82.

[0103]In a still more preferred embodiment, said process is carried out
with at least one primer selected from the group consisting of:
[0104]SEQ ID NO: 9, 12, 14, 15;and with at least one primer selected from
the group consisting of: [0105]SEQ ID NO: 24, 52, 82;and, optionally,
with at least one probe selected from the group consisting of: [0106]SEQ
ID NO: 91, 100, 109, 110, 118, 127, 136, and the complementary sequences
thereof.

wherein X indicates that the primers can be combined with each other as a
pair, and that each primer pair can in addition be used in combination
with any one of the probes selected from the group consisting of:
[0108]SEQ ID NO: 91, 100, 109, 110, 118, 127, 136, and the complementary
sequences thereof.

[0109]In another aspect of the invention, said reference template sequence
is SEQ ID NO: 4, from the L20587 HIV1-O reference isolate, or a fragment
of SEQ ID NO: 4 selected from 4336-4484, 4338-4484.

[0110]In one embodiment of the invention, said reference template sequence
is SEQ ID NO: 4, from the L20587 HIV1-O reference isolate or one of said
fragments of SEQ ID NO: 4, and said process is carried out with at least
one oligonucleotide selected from the group consisting of: [0111]SEQ ID
NO: 148, 157, 167, 170, 179, 189, 190 to 194, and the sequences which are
complementary to one of SEQ ID NO: 190 to 194 over the entire length of
this SEQ ID NO:

[0112]In a preferred embodiment, said process is carried out with at least
one primer selected from the group consisting of: [0113]SEQ ID NO: 148,
157, 167, 170, 179, 189.

[0114]In another embodiment, said process is carried out with at least one
probe selected from the group consisting of: [0115]SEQ ID NO: 190 to
194, and the complementary sequences thereof.

[0116]In a more preferred embodiment, said process is carried out with at
least one primer selected from the group consisting of: [0117]SEQ ID
NO: 148, 157, 167;and with at least one primer selected from the group
consisting of: [0118]SEQ ID NO: 170, 179, 189.

[0119]In a still more preferred embodiment, said process is carried out
with at least one primer selected from the group consisting of:
[0120]SEQ ID NO: 148, 157, 167;and with at least one primer selected from
the group consisting of: [0121]SEQ ID NO: 170, 179, 189;and, optionally,
with at least one probe selected from the group consisting of: [0122]SEQ
ID NO: 190 to 194, and the complementary sequences thereof.

wherein X indicates that the primers can be combined with each other as a
pair, and that each primer pair can in addition be used in combination
with any one of the probes selected from the group consisting of:
[0124]SEQ ID NO: 190, 191, 192, 193, 194, and the complementary sequences
thereof.

[0125]In another aspect of the invention, said reference template sequence
is SEQ ID NO: 5, from the M30502 HIV2 reference isolate.

[0126]In one embodiment of the invention, said reference template sequence
is SEQ ID NO: 5, from the M30502 HIV2 reference isolate, and said process
is carried out with at least one oligonucleotide selected from the group
consisting of: [0127]SEQ ID NO: 221, 249, 276, 287, 296, 297 to 303,
and the sequences which are complementary to one of SEQ ID NO: 297 to 303
over the entire length of this SEQ ID NO:

[0128]In a preferred embodiment, said process is carried out with at least
one primer selected from the group consisting of: [0129]SEQ ID NO: 221,
249, 276, 287, 296.

[0130]In another embodiment, said process is carried out with at least one
probe selected from the group consisting of: [0131]SEQ ID NO: 297 to
303, and the complementary sequences thereof.

[0132]In a more preferred embodiment, said process is carried out with at
least one primer selected from the group consisting of: [0133]SEQ ID
NO: 221, 249;and with at least one primer selected from the group
consisting of: [0134]SEQ ID NO: 276, 287, 296.

[0135]In a still more preferred embodiment, said process is carried out
with at least one primer selected from the group consisting of:
[0136]SEQ ID NO: 221, 249;and with at least one primer selected from the
group consisting of: [0137]SEQ ID NO: 276, 287, 296;and, optionally, with
at least one probe selected from the group consisting of: [0138]SEQ ID
NO: 297 to 303, and the complementary sequences thereof.

wherein X indicates that the primers can be combined with each other as a
pair, and that each primer pair can in addition be used in combination
with any one of the probes selected from the group consisting of:
[0140]SEQ ID NO: 297, 298, 299, 300, 301, 302, 303, and the complementary
sequences thereof.

[0141]In another aspect of the invention, said reference template sequence
is SEQ ID NO: 1, from the K03455 HIV1-M reference isolate, or the
fragment of SEQ ID NO:1 which is identical to positions 4176-4429 of said
K03455 HIV1-M reference isolate.

[0142]In one embodiment of the invention, said reference template sequence
is SEQ ID NO: 1, from the K03455 HIV1-M reference isolate, or said
fragment thereof, and said process is carried out with at least one
oligonucleotide selected from the group consisting of: [0143]SEQ ID NO:
6, 24, 52, 82, 91, 100, 109, 118, 127, 136, and the sequences which are
complementary to one of SEQ ID NO: 91, 100, 109, 118, 127, 136, over the
entire length of this SEQ ID NO:

[0144]In a preferred embodiment, said process is carried out with at least
one primer selected from the group consisting of: [0145]SEQ ID NO: 6,
24, 52, 82.

[0146]In another embodiment, said process is carried out with at least one
probe selected from the group consisting of: [0147]SEQ ID NO: 91, 100,
109, 118, 127, 136, and the complementary sequences thereof.

[0148]In a more preferred embodiment, said process is carried out with at
least one primer selected which is: [0149]SEQ ID NO: 6;and with at
least one primer selected from the group consisting of: [0150]SEQ ID NO:
24, 52, 82.

[0151]In a still more preferred embodiment, said process is carried out
with at least one primer selected which is: [0152]SEQ ID NO: 6;and with
at least one primer selected from the group consisting of: [0153]SEQ ID
NO: 24, 52, 82;and, optionally, with at least one probe selected from the
group consisting of: [0154]SEQ ID NO: 91, 100, 109, 118, 127, 136, and
the complementary sequences thereof.

wherein X indicates that the primers can be combined with each other as a
pair, and that each primer pair can in addition be used in combination
with any one of the probes selected from the group consisting of:
[0156]SEQ ID NO: 91, 100, 109, 118, 127, 136, and the complementary
sequences thereof.

[0157]In another aspect of the invention, said reference template sequence
is SEQ ID NO: 3, from the L20587 HIV1-O reference isolate, or the
fragment of SEQ ID NO:3 which is identical to positions 4231-4484 of said
L20587 HIV1-O reference isolate.

[0158]In one embodiment of the invention, said reference template sequence
is SEQ ID NO: 3, from the L20587 HIV1-O reference isolate, or said
fragment thereof, and said process is carried out with at least one
oligonucleotide selected from the group consisting of: [0159]SEQ ID NO:
139, 170, 179, 189, 190 to 194, and the sequences which are complementary
to one of SEQ ID NO: 190 to 194 over the entire length of this SEQ ID NO:

[0160]In a preferred embodiment, said process is carried out with at least
one primer selected from the group consisting of: [0161]SEQ ID NO: 139,
170, 179, 189.

[0162]In another embodiment, said process is carried out with at least one
probe selected from the group consisting of: [0163]SEQ ID NO: 190 to
194, and the complementary sequences thereof.

[0164]In a more preferred embodiment, said process is carried out with at
least one primer which is: [0165]SEQ ID NO: 139;and with at least one
primer selected from the group consisting of: [0166]SEQ ID NO: 170, 179,
189.

[0167]In a still more preferred embodiment, said process is carried out
with at least one primer which is: [0168]SEQ ID NO: 139;and with at
least one primer selected from the group consisting of: [0169]SEQ ID NO:
170, 179, 189;and, optionally, with at least one probe selected from the
group consisting of: [0170]SEQ ID NO: 190 to 194, and the complementary
sequences thereof.

wherein X indicates that the primers can be combined with each other as a
pair, and that each primer pair can in addition be used in combination
with any one of the probes selected from the group consisting of:
[0172]SEQ ID NO: 190, 191, 192, 193, 194, and the complementary sequences
thereof.

[0173]In one aspect of the invention, said step of producing at least one
amplicon comprises at least one quantitative and/or qualitative,
multiplex and/or simplex PCR amplification.

[0174]In another aspect of the invention, said step of detecting
preferentially includes real-time and/or quantitative and/or end-point
detection.

[0175]In another aspect, the present invention provides an amplicon
obtainable by the above-described process, i.e. by implementation of the
process of the invention on a HIV-containing sample.

[0176]In a further aspect, there is provided an amplification composition
comprising at least one amplicon according to the invention. By
amplification composition, we herein understand any composition
obtainable by amplification, especially by PCR.

[0177]The invention is also directed to a polynucleotide suitable for use
as a reference template sequence in the design of primers that can be
used in multiplex to cover at least HIV1-M, HIV1-0, HIV2-A and HIV2-B in
a single amplification run while still offering a real time quantitative
amplification thereof. Naturally, the polynucleotides according to the
present invention are also suitable for further protocols, including
simplex protocols, multiplex protocols, end-point protocols, qualitative
protocols, quantitative protocols, combinations thereof, and the like.

[0178]By polynucleotide, we hereby understand any polymer of nucleotides,
wherein nucleotides can be ribonucleotides, deoxyribonucleotides,
dideoxyribonucleotides, degenerated nucleotides, and the like. Said
nucleotides are preferably single-stranded, but can also be double
stranded. The length of said polynucleotides can vary, and is usually
under 500 nucleotides (nt), preferably in the range of 50-400 nt, more
preferably 100-300 nt, even more preferably 150-250 nt.

[0186]In a further embodiment, said reference template polynucleotide is:
[0187]positions 4231-4491 (SEQ ID NO: 3) of the L20587 HIV1-O reference
isolate (see FIG. 7), or the fragment of SEQ ID NO:3 which is identical
to positions 4231-4484 of said L20587 HIV1-β isolate.

[0188]According to the present invention, there is provided an
oligonucleotide suitable for HIV detection: see FIGS. 7 to 21.

[0189]In one embodiment, the invention provides an oligonucleotide which
is selected from the group consisting in: [0190]SEQ ID NO: 6 to 304,
and the sequences which are complementary to one of SEQ ID NO: 83-138,
190-194, 297-304 over the entire length of this SEQ ID NO:

[0194]In one aspect, the invention provides an oligonucleotide which is
suitable for HIV1-M detection, and which is selected from the group
consisting in: [0195]SEQ ID NO: 6, 9, 12, 14, 15, 24, 52, 82, 91, 100,
109, 118, 127, 136, and the sequences which are complementary to one of
SEQ ID NO: 91, 100, 109, 118, 127, 136 over the entire length of this SEQ
ID NO.

[0196]In another aspect, the invention provides an oligonucleotide which
is suitable for HIV1-0 detection and which is selected from the group
consisting in: [0197]SEQ ID NO: 139, 148, 157, 167, 170, 179, 189, 190
to 194, and the sequences which are complementary to one of SEQ ID NO:
190-194, over the entire length of this SEQ ID NO.

[0198]In yet another aspect, the invention provides an oligonucleotide
which is suitable for HIV2-A detection and which is selected from the
group consisting in: [0199]SEQ ID NO: 221, 249, 276, 287, 296, 297 to
299, and the sequences which are complementary to one of SEQ ID NO:
297-299, over the entire length of this SEQ ID NO.

[0200]In yet another aspect, the invention provides an oligonucleotide
which is suitable for HIV2-B detection and which is selected from the
group consisting in: [0201]SEQ ID NO: 221, 249, 276, 287, 296, 300 to
303, and the sequences which are complementary to one of SEQ ID NO:
300-303, over the entire length of this SEQ ID NO:

wherein X indicates that the oligonucleotides can be combined with each
other as a pair (primer pair), and that each primer pair can in addition
be used in combination with any one of the oligonucleotides (probes)
selected from the group consisting of: [0214]SEQ ID NO: 91, 100, 109,
110, 118, 127, 136, and the complementary sequences thereof.

[0215]In a further aspect, the invention provides a set of
oligonucleotides suitable for HIV1-O detection.

[0216]In a preferred embodiment, the invention provides a set of
oligonucleotides suitable for HIV1-0 detection, comprising: [0217]at
least one oligonucleotide (primer) selected from the group consisting in
SEQ ID NO: 139, 148, 157, 167; and/or [0218]at least one oligonucleotide
(primer) selected from the group consisting in SEQ ID NO: 170, 179, 189;
and/or [0219]at least one oligonucleotide (probe) selected from the group
consisting in SEQ ID NO: 190 to 194, and the sequences which are
complementary to one of SEQ ID NO: 190-194, over the entire length of
this SEQ ID NO.

[0220]The possible combinations of oligonucleotides are thus as follows:

wherein X indicates that the oligonucleotides can be combined with each
other as a pair (primer pair), and that each primer pair can in addition
be used in combination with any one of the oligonucleotides (probes)
selected from the group consisting of: [0221]SEQ ID NO: 190, 191, 192,
193, 194, and the complementary sequences thereof.

[0222]In a further aspect, the invention provides a set of
oligonucleotides suitable for HIV2-A detection.

[0223]In a preferred embodiment, there is provided a set of
oligonucleotides suitable for HIV2-A detection, comprising: [0224]at
least one oligonucleotide (primer) selected from the group consisting in
SEQ ID NO: 221, 249; and/or [0225]at least one oligonucleotide (primer)
selected from the group consisting in SEQ ID NO: 276, 287, 296; and/or.
[0226]at least one oligonucleotide (probe) selected from the group
consisting in SEQ ID NO: 297 to 299 and the sequences which are
complementary to one of SEQ ID NO: 297-299 other the entire length of
this SEQ ID NO.

[0227]The possible combinations of oligonucleotides are thus as follows:

TABLE-US-00012
SEQ ID NO: 221 249
276 X X
287 X X
296 X X

wherein X indicates that the oligonucleotides can be combined with each
other as a pair (primer pair), and that each primer pair can in addition
be used in combination with any one of the oligonucleotides (probes)
selected from the group consisting of: [0228]SEQ ID NO: 297, 298, 299
and the complementary sequence thereof.

[0229]In a further aspect, the invention provides a set of
oligonucleotides suitable for HIV2-B detection.

[0230]In a preferred embodiment, there is provided a set of
oligonucleotides suitable for HIV2-B detection, comprising: [0231]at
least one oligonucleotide (primer) selected from the group consisting in
SEQ ID NO: 221, 249; and/or [0232]at least one oligonucleotide (primer)
selected from the group consisting in SEQ ID NO: 276, 287, 296; and/or
[0233]at least one oligonucleotide (probe) selected from the group
consisting in SEQ ID NO: 300 to 303, and the sequences which are
complementary to one of SEQ ID NO: 300-303, over the entire length of
this SEQ ID NO.

[0234]The possible combinations of oligonucleotides are thus as follows:

TABLE-US-00013
SEQ ID NO: 221 249
276 X X
287 X X
296 X X

wherein X indicates that the oligonucleotides can be combined with each
other as a pair (primer pair), and that each primer pair can in addition
be used in combination with any one of the oligonucleotides (probes)
selected from the group consisting of: [0235]SEQ ID NO: 300, 301, 302,
303, and the complementary sequences thereof.

[0236]In a further aspect, the invention provides a set of
oligonucleotides suitable for HIV multiplex detection.

[0237]In a preferred embodiment, there is provided a set of
oligonucleotides suitable for HIV detection, preferably multiplex
detection, comprising: [0238]at least one oligonucleotide selected from
the group consisting in SEQ ID NO: 91, 100, 109, 118, 127, 136, and the
complementary sequences thereof; and/or [0239]at least one
oligonucleotide selected from the group consisting in SEQ ID NO: 190 to
194, and the complementary sequences thereof; and/or [0240]at least one
oligonucleotide selected from the group consisting in SEQ ID NO: 297 to
299, and the complementary sequences thereof; and/or [0241]at least one
oligonucleotide selected from the group consisting in SEQ ID NO: 300 to
303, and the complementary sequences thereof; and/or [0242]combinations
thereof.

[0243]Such a set of oligonucleotides according to the invention may thus
comprise any combination thereof of 2, 3, 4, 5 and more of said
oligonucleotides.

[0244]In a preferred embodiment, such a set of oligonucleotides can be a
combination of probes, e.g. a combination of an HIV1-M probe and an
HIV1-O probe:

[0247]In a preferred embodiment, in any set of oligonucleotides according
to the invention, at least one of said oligonucleotides is fluorescently
labelled.

[0248]There is further provided an amplicon obtainable by means of at
least one oligonucleotide according to the invention, and/or at least one
set of nucleotides according to the invention.

[0249]There is more particularly provided an amplicon obtainable by
amplification from a HIV-containing sample with a pair of primers
selected from: [0250]a primer of SEQ ID NO: 9, 12, 14 or 15, and a
primer of SEQ ID NO: 24, 52 or 82; or [0251]a primer of SEQ ID NO: 148,
157 or 167, and a primer of SEQ ID NO: 170, 179 or 189; or [0252]a primer
of SEQ ID NO: 221 or 249, and a primer of SEQ ID NO: 276, 287 or 296; or
[0253]a primer of SEQ ID NO: 6, and a primer of SEQ ID NO: 24, 52 or 82;
or [0254]a primer of SEQ ID NO: 139, and a primer of SEQ ID NO: 170, 179
or 189.

[0255]There is also provided an amplicon which has a nucleotide length
identical to the nucleotide length of one of the reference template
sequences of the invention (i.e. SEQ ID NO: 2, 4, 5, 1 or 3 and the
above-mentioned fragments thereof), and which comprises a sequence having
a percentage of nucleotide identity of at least 90%, preferably of at
least 91%, most preferably of at least 92%, most preferably of at least
93%, most preferably of at least 94%, most preferably of at least 95%,
most preferably of at least 96%, most preferably of at least 97%, most
preferably of at least 98%, most preferably of at least 99%, most
preferably of 100%, with a probe sequence, over the entire length of this
probe sequence, wherein this probe sequence is one of the following: SEQ
ID NO: 91, 100, 109, 118, 127, 136, 190, 191, 192, 193, 194, 297, 298,
299, 300, 301, 302, 303, and the sequences which are complementary to
these SEQ ID NO: over the entire length of these SEQ ID NO.

[0256]An amplification composition comprising such an amplicon is also
encompassed by the present invention.

[0257]It is another object of the present invention to provide with a kit.

[0258]In a preferred embodiment, said kit comprises at least one
oligonucleotide (primer or probe) according to the invention, as
described above.

[0259]In another embodiment, said kit comprises at least one primer pair
according to the invention, as described above.

[0260]In yet another embodiment, said kit comprises at least a set of
oligonucleotides according to the invention, for example at least one
plurality of probes according to the invention, as described above.

[0261]In the kit according to the invention, the oligonucleotides
(primers, probes) can be either kept separately, or partially mixed, or
totally mixed.

[0262]Said oligonucleotides can be provided under dry form, or solubilized
in a suitable solvent, as judged by the skilled person. Suitable solvents
include TE, PCR-grade water, and the like.

[0263]In a preferred embodiment, the kit according to the invention can
also contain further reagents suitable for a PCR step, possibly including
reagents suitable for an RT-PCR step.

[0264]Such reagents are known to those skilled in the art, and include
water, like nuclease-free water, RNase free water, DNAse-free water,
PCR-grade water; salts, like magnesium, potassium; buffers such as Tris;
enzymes, including polymerases, such as Taq, Vent, Pfu (all of them
Trade-Marks), activable polymerase, reverse transcriptase, and the like;
nucleotides like deoxynucleotides, dideoxunucleotides, dNTPs, dATP, dTTP,
dCTP, dGTP, dUTP; other reagents, like DTT and/or RNase inhibitors; and
polynucleotides like polyT, polydT, and other oligonucleotides, e.g.
primers.

[0265]In another preferred embodiment, the kit according to the invention
comprises PCR controls. Such controls are known in the art, and include
qualitative controls, positive controls, negative controls, internal
controls, quantitative controls, internal quantitative controls, as well
as calibration ranges. The internal control for said PCR step can be a
template which is unrelated to the target template in the PCR step. Such
controls also may comprise control primers and/or control probes. For
example, in the case of HIV detection, it is possible to use as an
internal control, a polynucleotide chosen within a gene whose presence is
excluded in a sample originating from a human body (for example, from a
plant gene), and whose size and GC content is equivalent to those from
the target sequence.

[0266]In a preferred embodiment, the kit according to the invention
contains means for extracting and/or purifying nucleic acid from a
biological sample, e.g. from blood, serum, plasma. Such means are well
known to those skilled in the art. In a preferred embodiment, the kit
according to the invention contains instructions for the use thereof.
Said instructions can advantageously be a leaflet, a card, or the like.
Said instructions can also be present under two forms: a detailed one,
gathering exhaustive information about the kit and the use thereof,
possibly also including literature data; and a quick-guide form or a
memo, e.g. in the shape of a card, gathering the essential information
needed for the use thereof.

[0267]In a preferred embodiment, said kit is a diagnostics kit, especially
an in vitro diagnostics kit, i.e. an HIV diagnostics kit.

[0268]The present invention also relates to the field of diagnostics. The
oligonucleotides according to the present invention, and as described
above, can be used for the in vitro diagnostics of HIV types and
subtypes. In particular, the primers and probes according to the
invention can be used for in vitro typing, sub-typing, and quantification
of HIV nucleic acids present in an in vitro sample, for instance, in a
patient's blood, plasma and/or serum, or in a cell culture supernatant.

[0269]It is also an object of the present invention to provide with a
method to detect HIV nucleic acid presence in a sample.

[0270]In one embodiment, said method comprises the step of providing with
at least one sample suspected of comprising at least one target template
from at least one HIV type and/or sub-type and/or isolate.

[0271]By nucleic acid, we hereby understand any nucleic acid: it can be
synthetic or not, recombinant or naturally occurring, linear or circular.
This includes DNA and RNA. The nucleic acid can be either single stranded
or double stranded or even triple stranded. It can stem from various
biological sources, such as micro organisms (bacteria, yeasts, and the
like), or higher organisms, like mammal cells. Said nucleic acid can also
be of viral nature, e.g. retroviral nature, like HIV's. The nucleic acid
can also comprise total DNA, total RNA, genomic DNA, mitochondrial DNA,
plasmidic DNA, BAC DNA, and mixtures thereof. Moreover, the nucleic acid
can assume various states of purity.

[0272]By sample, we hereby understand any kind of sample, naturally
occurring or not. Preferably, said sample is from biological origin. Said
sample may also stem from a cell culture supernatant. In a preferred
embodiment, said sample derives from blood. More preferably, said nucleic
acid containing sample derives from serum and/or plasma. Said sample
might also result from a preliminary step. For instance, said sample
might be obtainable via a purification and/or extraction procedure, e.g.
from a blood sample. In particular, said sample may result from a
separation and/or purification and/or extraction process carried out on a
biological sample. Said sample can also be a control sample. Control
samples include samples as qualitative control, positive control,
negative control, quantitative control, and calibrating control. Said
control can be internal as well as external. Any sample according to the
present invention can be present several times. For instance, said sample
can be provided as duplicate, as triplicate, as quadruplicate . . . as
multiplicate, which is advantageous in the case of quantitative
experiments.

[0273]The skilled person is familiar with the notion of target template.
Said target template can be any nucleic acid, whose presence is to be
assessed in said method. Said target template is possibly, but not
necessarily, the nucleic acid to be amplified in said method (PCR
amplicon).

[0274]Said method may comprise the step of providing with at least one
nucleic acid-containing sample suspected of comprising at least one
target template from at least one HIV type and/or sub-type.

[0275]In one aspect of the invention, said method comprises the step of
providing with at least one oligonucleotide according to the invention
(e.g. PCR primer and/or probe) and/or at least one set of nucleotides
(e.g. a primer pair) according to the invention.

[0276]In one embodiment, said method comprises the step of contacting said
nucleic acid-containing sample with at least one oligonucleotide
according to the invention and/or at least one set of nucleotides
according to the invention, under conditions enabling the annealing of
said primer and/or said primer pair and/or said probe onto said template.

[0277]In another aspect, said method comprises the step of observing or
detecting the presence of an annealed product, thereby revealing the
initial presence of an HIV nucleic acid in said sample.

[0278]It is a further object of the invention to provide with a method for
the quantitative specific detection of HIV types and sub-types in a
sample, preferably through real-time quantitative multiplex PCR analysis.

[0279]In one embodiment, said method comprises the step of providing with
at least one sample suspected of comprising at least one target template
from at least one HIV type and/or sub-type.

[0280]In another preferred embodiment, said method comprises the step of
providing with at least one oligonucleotide according to the invention
(e.g. PCR primer and/or probe) and/or at least one set of nucleotides
according to the invention (e.g. primer pair).

[0281]In a preferred embodiment, said probe is suitable for the detection
of a putative HIV amplicon obtainable with said primer pair.

[0282]In a preferred embodiment according to the invention, spectral
properties of said probes can be chosen as to not interfere with each
other. In particular, when probes are used in multiplex, each single
probe can have its own fluorophore being spectrally significantly
different from each other, i.e. the absorption/emission spectra are
essentially non-overlapping. This advantageously allows for low-noise
multiplex detection for all single probes, making sure that individual
signals do not interfere with each other in detection. Examples of dyes
which can be used together in multiplex include Fam with Tamra, Fam with
Tamra and Texas Red.

[0283]In another preferred embodiment, said method comprises the step of
contacting said sample, in presence of said oligonucleotide(s) and/or
primer(s) and/or pair(s) or primers and/or probe(s) and/or set(s) of
oligonucleotides and possibly in the presence of suitable reagents, to
the conditions suitable for the PCR amplification of said target template
with said primer pair(s) and/or set(s).

[0284]Said PCR amplification can be any PCR reaction, including RT-PCR.
Such suitable reagents are known in the art, and examples thereof include
water, like nuclease-free water, RNase free water, DNAse-free water,
PCR-grade water; salts, like magnesium, potassium; buffers such as Tris;
enzymes, including polymerases, such as Taq, Vent, Pfu (all of them
Trade-Marks), activable polymerase, reverse transcriptase, and the like;
nucleotides like deoxynucleotides, dideoxynucleotides, dNTPs, dATP, dTTP,
dUTP, dCTP, dGTP; other reagents, like DTT and/or RNase inhibitors; and
polynucleotides like polyT, polydT. Advantageously according to the
invention, at least part of these reagents can be used as a pre-mix. The
amounts thereof to be used are known to those skilled in the art.

[0285]In a preferred embodiment, the primers according to the invention
are used in a final concentration range 100-200 nM. Typically, said
primers can be used at a final concentration range 200-1500 nM,
preferably 250-1000 nM, more preferably 500-1000 nM, even more preferably
600-1000 nM.

[0286]Probe concentration in a PCR reaction can be optimized, typically by
varying the final concentration from 50 nM to 1000 nM. In a preferred
embodiment, the probes according to the invention are used at a final
concentration range 50-1000 nM, preferably 100-800 nM, more preferably
100-600 nM, even more preferably 200-600 nM.

[0287]Said conditions are known to those skilled in the art. They include
temperature conditions, in particular thermal cycling conditions, e.g.
temperature, duration, number, heating rate of the cycles. In a preferred
embodiment, said temperature conditions include conditions suitable for
an RT-PCR. In another preferred embodiment, said conditions include
conditions suitable for a QPCR. In yet another preferred embodiment, said
conditions include conditions suitable for a quantitative RT-PCR.

[0288]In another embodiment, said method comprises the step of bringing
said sample, in the presence of said probe(s) under conditions suitable
for the annealing of said probe to said putative amplicon.

[0289]In yet another preferred embodiment, said method comprises the step
of detecting at least once, preferably real-time, potential amplification
products, i.e. whether said probe(s) meet(s) said amplicon to anneal
with, preferably for each sample. This advantageously allows for the
assessment of the presence of said HIV type and/or sub-type. This can be
advantageously achieved by fluorescence intensity measurements.
Fluorescence measurement procedures are known in the art. Briefly, the
sample is illuminated at around the excitation wavelength of the
fluorophore, and emission intensity is measured.

[0290]In another embodiment, said method comprises the step of measuring
at least once, preferably real-time, the amount of said probe annealed to
said amplicon. This can be advantageously achieved by fluorescence
intensity measurements. Fluorescence measurement procedures are known in
the art. Briefly, the sample is illuminated at around the excitation
wavelength of the fluorophore, and emission intensity is measured

[0291]In another preferred embodiment, said method comprises the step of
estimating at least once the number of target template copies initially
present in said sample. The skilled person would know how to carry out
such a step. For example, this can be advantageously performed having
used calibration standards and/or internal controls. Preferably, this
step includes the determination of the so-called threshold cycle (CT) for
each sample, which correlates to the number of target template copies
initially present in said sample.

[0292]In a preferred embodiment, at least one step, preferably several
steps, more preferably most of the steps of said method can be carried
out in a PCR plate. Suitable such PCR plates are known in the art. They
include 24-well plates, 48-well plates, 96-well-plates, and 284-well
plates. This advantageously ensures that samples can be processed in
parallel in said steps. In addition, this allows for high throughput
screening, which advantageously saves time. In another preferred
embodiment, at least one step, preferably several steps, more preferably
most steps of said method can be carried out in a thermal cycler. Such
thermal cycler might be equipped for real-time fluorescence intensity
measurements, in which case said plates can advantageously be of
optical-grade.

[0293]This application also relates to the amplification of HIV nucleic
acids with at least one oligonucleotide and/or PCR primer and/or at least
one PCR primer pair and/or at least one probe and/or at least one set of
nucleotides according to the invention.

[0294]The skilled person can appreciate that the present invention can
incorporate any number of the preferred features described above. All
citations mentioned herein are hereby incorporated by reference in their
entirety.

[0295]Other embodiments of the present invention are not presented here,
which are obvious to those skilled in the art, and thus are within the
scope and the spirit of the present invention. In particular, although
being suitable for detection via multiplex and/or real-time protocols,
the methods, processes, polynucleotides, oligonucleotides, sets of
oligonucleotides, amplicons, and kits of the present invention are
obviously also suitable for simplex protocols, qualitative protocols,
quantitative protocols, end-point detection protocols, and combinations
thereof.

[0296]The advantages of the products, processes and methods according to
the invention will become apparent from the following examples, which are
given below as mere illustrations, and are non limitative.

[0300]FIGS. 6A, 6B and 6C illustrate an HIV2/internal control assay
according to the present invention (see example 5 below).

[0301]FIGS. 7 to 21 illustrate sequences according to the present
invention.

EXAMPLES

Example 1

HIV2 detection (sub-types A and B) (FIGS. 1A, 1B, 2A, 2B and 3)

[0302]This example illustrates that the HIV2 primers and probes of the
invention allow for RT-PCR specific real-time detection of HIV2 sub-types
A and B. This example involves the use of one pair of HIV2 primers, and
two different HIV2 probes. One probe is an HIV2-A probe (sub-type A
probe), and the other one is an HIV2-B probe (sub-type B probe). These
primers and probes target a 147 bp sequence in HIV2 isolates.

[0305]Each of these probes is used as a molecular beacon in this example.
The target-unrelated beacon arms which have been added at each end of
each probe are shown underlined (FAM=fluorophore; DQ=Dark Quencher).

[0321]The panel of sub-type A samples and sub-type B samples are submitted
to nucleic acid extraction and RT-PCR amplification using the HIV2 primer
pair together with either only one of the probes (experiments a) and b)),
or both probes (experiment c)).

[0322]Interpretation of the results: for each assay, one determines a
threshold cycle (Ct) which is the level of fluorescence that is
considered to be significantly above the background level of fluorescence
measured in the early cycles of the amplification. The Ct value is
inversely proportional to the concentration of target: the lower the Ct,
the higher the concentration of target.

[0323]In the following tables, CT=Threshold Cycle; RFU max=maximal
Relative Fluorescence Units observed at the end of the PCR run;
CTL-=negative control; N/A=sample whose level of fluorescence is below
the background level.

[0329]As a remarkable feature of the invention, the HIV2-A probe (SH2A9a,
SEQ ID NO: 298) and the HIV2-B probe (SH2A1b, SEQ ID NO: 301) can be used
simultaneously (see Table 3 and FIG. 3) and allow for detection of both
sub-types in a one-step multiplex (here duplex) procedure.

[0330]As a surprising feature, a synergistic effect is observed for the
simultaneous use of both A and B probes (sub-type A probe and sub-type B
probe) (duplex protocol), compared to the use of a single probe alone
(sub-type A probe or sub-type B probe). The detection signal obtained
with a single probe is slightly less accurate than the respectively
obtained with both probes, as can be judged from the comparison of the
results obtained in experiment a) with those obtained in experiment c) on
sub-type A samples, and from the comparison of the results obtained in
experiment b) with those obtained on sub-type B samples in experiment c).

[0331]The sets of oligonucleotides (primers and probes) according to the
invention thus prove an increased specificity when probes are used
simultaneously (multiplex, duplex protocol), as a surprising and
unexpected synergistic effect

[0335]According to the invention, each probe is produced here as a
molecular beacon, with "TGCGC" as target-unrelated arm in 5', and with
"GCGCA" as target-unrelated arm in 3' (both arms are underlined). The
5'-arm is labelled with a FAM fluorophore, and the 3' arm with a Dark
Quencher.

[0350]The same RT-PCR procedure has been followed for both types of
amplification experiments.

[0351]Interpretation of the results: for each assay, one determines a
threshold cycle (Ct) which is the level of fluorescence that is
considered to be significantly above the background level of fluorescence
measured in the early cycles of the amplification. The Ct value is
inversely proportional to the concentration of target: the lower the Ct,
the higher the concentration of target.

[0352]In the following tables:

CT=Threshold Cycle;

[0353]RFU max=maximal Relative Fluorescence Units observed at the end of
the PCR run;CTL-=negative control;N/A=sample whose level of fluorescence
is below the background level;Avg.: average.

[0356]On FIG. 4A, are shown the results obtained with the HIV1-M primers
(H1B4f, H1B10r, SEQ ID NO: 7, 16) and HIV1-M probes (SH1BM5, SEQ ID NO:
119) of the invention on the HIV1-M panel (BBI PRD201), on the HIV1-O
panel (BBI PRD301), and on the controls.

[0357]On FIG. 4B, are shown the results obtained with the HIV 1-O primers
(H1B5f, H1B13r, SEQ ID NO: 140, 168) and HIV1-O probes (SH1BO2, SEQ ID
NO: 191) of the invention on the HIV1-0 panel, on the HIV1-M panel, and
on the controls.

[0358]In both cases, no amplification is detected in quantitative
real-time RT-PCR when the HIV1-M primers and probes of the invention are
used on HIV1-0 samples, and conversely.

[0359]Advantageously according to the invention, no cross-hybridization
occurs between the HIV1-M amplicon and the HIV1-O probes, nor between the
HIV1-O amplicon and the HIV1-M probes.

[0360]The HIV1-M and HIV 1-O primers and probes according to the invention
have thus proven to allow for very high specificity detection in
real-time quantitative RT-PCR conditions.

Example 3

HIV1-M and HIV1-O Quantification

[0361]This example illustrates that the primers and probes of the
invention allows for quantification of HIV viral charge by RT-PCR.

[0363]According to the invention, each probe is in this case produced as a
molecular beacon, with "TGCGC" as target-unrelated arm in 5', and with
"GCGCA" as target-unrelated arm in 3' (both arms are underlined). The 5'
arm is labelled with a FAM fluorophore, and the 3' arm with a Dark
Quencher.

[0383]The Ct results of each assay are used for the quantification of
panels PRD201 and PRD301 by mapping the corresponding Ct to the standard
curve. The quantification values corresponding to the number of RNA
copies/ml in each pure sample are compared with commercially-available
kits used in accordance with the manufacturers' recommendations: kit
Amplicor HIV1 Monitor Version 1.5 from Roche (ref 87674), kit Quantiplex
HIV1 RNA 3.0 bDNA from Bayer (ref 6147) and kit Nuclisens HIV-1 QT from
Organon Teknika (ref 84152).

[0385]It is apparent from these results that the HIV1-M primers and probes
of the invention allow for an accurate real-time quantitative detection
of all HIV1-M genotypes, and good correlation with the commercially
available kits.

[0386]It can be seen from Table 6 that the higher level of quantification
on HIV1-0 panel is obtained with both HIV1-M and HIV1-O primers and
probes of the invention. (<LDL=less than lower detection limit).

Example 4

HIV1/HIV2 Multiplex Assay (FIGS. 5A and 5B)

[0387]This example illustrates that the HIV1 and HIV2 primers and probes
of the invention may be used in a multiplex assay for HIV1 or HIV2
signal. It also demonstrates the possibility to follow the fluorescence
of two targets in the same tube by use of two different fluorophores (FAM
and ROX).

[0389]According to the invention, each probe is in this case produced as a
molecular beacon, with "CGCGC" as target-unrelated arm in 5', and with
"GCGCG" as target-unrelated arm in 3' (both arms are underlined). The 5'
arm is labelled with a FAM or ROX fluorophore, and the 3' arm with a
Dabcyl moiety.

[0404]Two series of amplification experiments have been performed with the
primers and probes of the invention: [0405]a) RT-PCR with a single pair
of primers and probe HIV1-M (H1B4f, H1B10r, SH1BM10, SEQ ID NO: 7, 16,
119, a single pair of primers and probe HIV2 (H2A3f, H2A3r, SH2A14a, SEQ
ID NO: 195, 250, 299) with a range of HIV1-M target concentrations.
[0406]b) RT-PCR with a single pair of primers and probe HIV1-M (H1B4f,
H1B10r, SH1BM10, SEQ ID NO: 7, 16, 119), a single pair of primers and
probe HIV2 (H2A3f, H2A3r, SH2A14a, SEQ ID NO: 195, 250, 299) with a range
of HIV2 target dilutions.

[0407]Interpretation of the results: for each assay one determines a
threshold cycle (Ct) which is the level of fluorescence that is
considered to be significantly above the background level of fluorescence
measured in the early cycles of the amplification. The Ct value is
inversely proportional to the concentration of target: the lower the Ct,
the higher the concentration of target.

[0408]In the following tables, CT=Threshold Cycle; RFU max=maximal
Relative Fluorescence Units observed at the end of the PCR run;
CTL-=negative control; N/A=sample whose level of fluorescence is below
the background level; Avg=average; cop=copies.

[0409]Its is apparent from these results that the HIV1 and HIV2 primers
and probe of the invention may be used in a multiplex assay for the
detection of HIV1-M or HIV2 target.

[0410]The results are illustrated by FIGS. 5A and 5B (for each dilution,
there are 2 curves since experiments are here carried out in duplicate).

Example 5

HIV2/IC Multiplex Assay (FIGS. 6A and 6B)

[0411]This example illustrates that the HIV2 target and internal control
(IC) may be co-amplified using the HIV2 primers and probes of the
invention and selected IC primers and probe without any perturbation of
the HIV2 signal. It also demonstrates the possibility to follow the
fluorescence of two targets in the same tube by use of two different
fluorophores (ROX and TAMRA)

[0412]This example involves the use of one pair of HIV2 primers and one
HIV2 probe. These primers and probes target a 147 bp sequence in HIV2
isolates. They have the following sequences:

[0413]This example also involves the use of one pair of IC primers and one
IC probe, selected to get the same amplified fragment size and GC % than
the HIV target.

[0414]Each of these probes is used as a molecular beacon in this example.
The target-unrelated beacon arms which have been added at each end of the
HIV2 probe is shown underlined (ROX=fluorophore; Dabcyl=quencher).

[0431]HIV2-A positive sample was submitted to nucleic acid extraction and,
after dilution to 1/300, 1/3000 and 1/30000 in water to RT-PCR
amplification as described in experiments a) and b).

[0432]IC dilution was submitted to RT-PCR amplification (around 106
cop/PCR) as described in experiment b). (cop=copies).

[0433]Interpretation of results: for each assay is determined a threshold
cycle (Ct) which is the level of fluorescence that is considered to be
significantly above the background level of fluorescence measured in the
early cycles of the amplification. The Ct value is inversely proportional
to the concentration of target: the lower the Ct, the higher the
concentration of target.

[0434]In the following tables, CT=Threshold Cycle; max RFU=maximal
Relative Fluorescence Units observed at the end of the PCR run;
CTL-=negative control; N/A=sample whose level of fluorescence is below
the background level.

[0436]It can be seen from this table that the IC Ct values are very
reproducible irrespective of the dilution of HIV2-A extracted sample
added.

[0437]These results are illustrated by FIGS. 6A, 6B and 6C (for each
dilution of HIV2-A, there are 3 curves since experiments are here carried
out in triplicate in FIGS. 6A and 6B; there are 6 curves for the negative
control; and there are 9 curves for the IC, all with the same
concentration on FIG. 6C).